In our studies of the biology of HIV macrophage infections we ask the following two questions: What cellular genes are regulated by HIV infection of macrophages? What viral determinants contribute to macrophage tropism? The first question we have addressed by using PCR-based subtractive hybridization methods to make libraries of genes up or down regulated by HIV infection of normal human macrophages in tissue culture. We have isolated several novel, previously unreported genes specifically upregulated by HIV infection of macrophages when compared to LPS treated controls. We are now analyzing the regulation of these genes by HIV infection of T cells and are following their expression in AIDS patients. The second question we are addressing by using a new saturation mutagenesis method called "genomic footprinting". In this technique, retroviral integrase is used to scatter defined primer insertions randomly throughout the HIV genome, theoretically hitting every position. Paired with primers homologous to known locations in the HIV genome, the primers can be detected by PCR. When a library of mutants is subjected to PCR and the products displayed on a sequencing gel, a ladder is seen, each position representing one primer insertion. When the library is passed through a biological or selective system and redisplayed, defective mutants drop out and are detected as bands missing from the ladder. We plan to make a library of HIV mutants in the dual tropic strain, DH12. We will passage this library through T cells and macrophages in parallel, running the displays side by side to detect differences.